PROJECT SUMMARY More than 98% of the human genome consists of noncoding sequences. The importance of these has been emphasized by genome-wide association studies (GWAS), which have identified many thousands of common genetic variants associated with human traits and disease susceptibility, the vast majority of which localize to the noncoding genome. In addition, as the cost of whole genome sequencing has dropped dramatically, clinical genomes have proliferated. A major bottleneck in realizing the potential of precision medicine and capitalizing on knowledge afforded by GWAS is the inability to understand and predict the functional consequences of perturbation of the noncoding genome. Up until recently, studies of the noncoding genome have been limited to ectopic heterologous reporter assays, correlative biochemical studies, or laborious knockout experiments in model organisms. Advances in genome editing have enabled facile disruption of human noncoding sequences in chromatinized cellular contexts. Recently we have developed a technique, Cas9-mediated in situ saturating mutagenesis, which allows the high-throughput and high-resolution perturbation of noncoding sequences. We hypothesize that only by perturbation in the appropriate chromatin and cellular environment can the requirement of noncoding sequences be established. In this proposal we describe comprehensive studies to characterize essential noncoding sequences required for erythropoiesis as marked by naturally occurring trait- associated genetic variation. Erythropoiesis is a particularly apt system to investigate noncoding genetic determinants given its predominantly cell-intrinsic nature, direct clinical relevance, and the availability of high- quality human genetic data, extensive chromatin maps, and faithful tissue culture models. With these studies, we will perturb trait-associated enhancers as well as non-enhancer noncoding elements to reveal minimal critical sequences required for erythropoiesis. We will introduce several technical advances, including utilization of alternative nucleases for pooled screening, haplotype-aware guide RNA design, predictions of on- target efficiency and off-target potential, and nuclease target deep sequencing, to approach nucleotide resolution determination of critical sequences. We will utilize bioinformatic, biochemical, and genome editing methods to define key trans-acting factors interacting with the essential cis-acting sequences. The overall goal will be to develop improved models of noncoding sequence function by iterative experimental testing and analytic refinement. These studies are intended to yield an improved understanding of blood cell development, identify novel rational targets for blood disorders, and illuminate fundamental mechanisms of gene regulation and trait heritability.